Synthesis of N-glycyl-β-glycopyranosyl amines,derivatives of natural oligosaccharides — glucose analogs of Lex antigen

Treatment of the natural tri-, tetra-, and pentasaccharides, β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, α-l-Fucp-(1→2)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, and α-l-Fucp-(1→2)-[α-d-GalNAcp-(1→3)]-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, which are glucose analogs of Le^x, with ammonium carbamate in aqueous methanol gave the corresponding β-glycopyranosyl amines. After their N-acylation with N-Z-glycine N-hydroxysuccinimidyl ester (Z is benzyloxycarbonyl) with subsequent hydrogenolytic removal of Z-group, corresponding N-glycyl-β-glycopyranosyl amines were obtained in yields up to 70%.     

Enantioselective Extraction System Containing Binary Chiral Selectors and Chromatographic Enantioseparation Method for Determination of the Absolute Configuration of Enantiomers of Cyclopentolate

The distribution coefficients and enantioseparation of cyclopentolate were studied in an extraction system containing d-tartaric acid ditertbutyl ester in organic phase and 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) in aqueous phase. Various parameters involved in the enantioseparation such as the type and the concentration of chiral selectors, pH value and a wide range of organic solvents were investigated. The maximum enantioselectivity (α = 2.13) and optimum distribution coefficients (K _R = 0.85, K _S = 0.40) were obtained under the following conditions: 0.10 mol/L HP-β-CD in aqueous phase and 0.20 mol/L d-tartaric acid ditertbutyl ester in decanol as organic phase. Cyclopentolate is present as a racemic mixture to the aqueous phase. The potentially different biological activities of cyclopentolate enantiomers have not been examined yet. Two chiral liquid chromatography methods have been developed for the direct separation of the enantiomers of cyclopentolate. First method was used for the quantification analysis of cyclopentolate enantiomers in aqueous phase. Second method used two chiroptical detectors: electronic circular dichroism (ECD) and optical rotation (OR) for the identification of individual cyclopentolate enantiomers from the organic phase enriched with (R)-enantiomer. The absolute stereochemistry was determined by means of the comparison of the experimental and computed ECD spectra and signs of OR. The ECD spectra of chiral analytes were measured on-line using HPLC-ECD technique.     

Solubility of l-tartaric Acid in Ethanol, Propanol, Isopropanol, n-Butanol, Acetone and Acetonitrile

The solubility of l-tartaric acid was measured in ethanol, propanol, isopropanol, n-butanol, acetone and acetonitrile in the temperature range 281.15 and 324.25 K under atmospheric pressure by a gravimetric method. The solubility of l-tartaric acid in those selected solvents increases with increasing temperature. The apparent molar enthalpies of solution of l-tartaric acid in the selected solvents were estimated from the solubility data. The solubility results were correlated with the van’t Hoff equation, the modified Apelblat equation, and the λh equation. Agreement with the experimental data was very good in all cases. The experimental results could be useful for optimizing the purification process of l-tartaric acid in industry.     

Study of the interaction between ethanol and natural amino acids containing ionic side groups in water at T = 298.15 K

The enthalpies of solution of l-α-aspartic acid, l-α-glutamic acid, l-α-arginine, l-α-lysine, and l-α-histidine have been measured in aqueous ethanol solutions at 298.15 K. From the obtained experimental results, the standard enthalpies of solution of amino acids in water–ethanol solutions have been determined. These data were used to calculate the heterogeneous enthalpic pair interaction coefficients based on McMillan–Mayer’s formalism. These values were interpreted in the terms of the ionic or no polar effect of the side chains of l-α-amino acids on their interactions with a molecule of ethanol in water.     

Neue enzymatische Analysenmethoden zur Bestimmung von Maltose,Stärke und Lactat in der Lebensmittelchemie

1. Determination of Maltose. Maltose is hydrolyzed by the enzyme α-glucosidase into glucose, which is determined by the enzymes hexokinase and glucose-6-phosphate-dehydrogenase. α-Glucosidase is specific for oligosaccharides with α-1,4 and α-1,2 bonds.
2. Determination of Starch and Glycogen. Starch and glycogen are splitted to glucose by the enzyme amylo-glucosidase. Starch has to be dissolved before enzymatic cleavage. A comparison of different methods for preparing starch solutions is given.
3. Determination of d- and l-Lactate. It is possible to determine d-lactate and l-lactate with the specific enzymes d-lactate-dehydrogenase and l-lactate-dehydrogenase. By different samples it is shown that no equal quantities of d- and l-lactate were found in the analyzed foods.

     

Enantiomer separation of phenyllactic acid by HPLC with Hp-β-cyclodextrin as chiral mobile phase additive

In our current research work, a sensitive, specific, and economic HPLC method has been developed for the separation of phenyllactic acid (PLA) enantiomers. The effects of cyclodextrin (CD) type, column temperature, buffer pH, methanol content and CD concentration on enantioselective separation were investigated. Baseline chromatographic separation was achieved on an Inertsil ODS-SP (150 × 4.6 mm, i.d. 5 μm) column with HP-β-CD as chiral mobile phase additive. The LOD and LOQ for d-phenyllactic acid were 0.3 and 0.7 μg/mL, respectively. A good linear relationship was obtained in the concentration range of 0.71–5.13 μg/mL with r² >0.999 for d-PLA. The percentage recovery of the d-PLA ranged from 94.1 to 103.2 in l-PLA. This method is simple and cost-saving, and could be easily applied for chiral discrimination and determination of the racemic PLA in an enantioselective study in quality control laboratory.     

The yjjN of E. coli codes for an l-galactonate dehydrogenase and can be used for quantification of l-galactonate and l-gulonate

Escherichia coli is able to utilize l-galactonate as a sole carbon source. A metabolic pathway for l-galactonate catabolism is described in E. coli, and it is known to be interconnected with d-galacturonate metabolism. The corresponding gene encoding the first enzyme in the l-galactonate pathway, l-galactonate-5-dehydrogenase, was suggested to be yjjN. However, l-galactonate dehydrogenase activity was never demonstrated with the yjjN gene product. Here, we show that YjjN is indeed an l-galactonate dehydrogenase having activity also for l-gulonate. The K _m and k _cat for l-galactonate were 19.5?±?0.6 mM and 0.51?±?0.03 s^?1, respectively. In addition, YjjN was applied for a quantitative detection of the both of these substances in a coupled assay. The detection limits for l-galactonate and l-gulonate were 1.65 and 10 μM, respectively.     

In vitro and in silico inhibition of angiotensin-converting enzyme by carbohydrates and cyclitols

Fifteen carbohydrates (d-mannose, d-glucose, d-galactose, methyl-α-d-glucose, l-rhamnose, d-xylose, d-fructose, d-arabinose, dulcitol, mannitol, β-maltose, α-lactose, melibiose, sucrose, and raffinose) and four cyclitols [l-(+)-bornesitol, myo-inositol, per-O-acetyl-1-l-(+)-bornesitol, and quinic acid] were assayed for in vitro ACE inhibition. Of these molecules, per-O-Acetyl-1-l-(+)-bornesitol, quinic acid, methyl-α-d-glucose, d-rhamnose, raffinose, and the disaccharides were determined to be either inactive or weak ACE inhibitors, whereas l-(+)-bornesitol, d-galactose, d-glucose, and myo-inositol exhibited significant ACE inhibition. Molecular docking studies were performed to investigate interactions between active compounds and human ACE (Protein Data Bank, PDB 1O83). The results of various calculations showed that all active sugars bind to the same enzyme region, which is a tunnel directed towards the active site. With the exception of myo-inositol (K _i = 13.95 μM, IC₅₀ = 449.2 μM), the active compounds presented similar K _i and IC₅₀ values. d-Galactose (K _i = 19.6 μM, IC₅₀ = 35.7 μM) and l-(+)-bornesitol (K _i = 25.3 μM, IC₅₀ = 41.4 μM) were the most active compounds, followed by d-glucose (K _i = 32.9 μM, IC₅₀ = 85.7 μM). Our docking calculations are in agreement with the experimental data and show a new binding region for sugar-like molecules, which may be explored for the development of new ACE inhibitors.     

Chiral separation and determination of excitatory amino acids in brain samples by CE-LIF using dual cyclodextrin system

Chiral capillary electrophoresis method has been developed to separate aspartate and glutamate enantiomers to investigate the putative neuromodulator function of d-Asp in the central nervous system. To achieve appropriate detection sensitivity fluorescent derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and laser-induced fluorescence detection was applied. Although, simultaneous baseline separation of the two enantiomer pairs could be achieved by using 3?mM 6-monodeoxy-6-mono(3-hydroxy)propylamino-β-cyclodextrin (HPA-β-CD), further improvement of the chemical selectivity was required because of the high excess of l-enantiomers in real samples to be analyzed. The system selectivity was fine-tuned by combination of 8?mM heptakis(2,6-di-O-methyl)-β-cyclodextrin and 5?mM HPA-β-CD in order to increase the resolution between aspartate and glutamate enantiomers. The method was validated for biological application. The limits of detection for d-Asp and d-Glu were 17 and 9?nM, respectively, while the limit of quantification for both analytes was 50?nM. This is the lowest quantification limit reported so far for NBD-tagged d-Asp and d-Glu obtained by validated capillary electrophoresis laser-induced fluorescence method. The applicability of the method was demonstrated by analyzing brain samples of 1-day-old chickens. In all the studied brain areas, the d-enantiomer contributed 1–2?% of the total aspartate content, corresponding to 17–45?nmol/g wet tissue.     

pH-dependent complex formation of amino acids with β-cyclodextrin and quaternary ammonium β-cyclodextrin

Stability constants for the complexes of anionic, neutral (zwitterionic) and protonated forms of l- and d-enantiomers of eight amino acids with β-cyclodextrin and the positively charged quaternary ammonium β-cyclodextrin (QA-β-CD, DS?=?3.6?±?0.3) have been determined by spectrophotometric and pH-potentiometric methods. The highest stability constants have been obtained for the aromatic amino acids phenylalanine, tyrosine and tryptophan. Except the dianion of tyrosine and QA-β-CD, values for the anions in the range of 80–120 have been found, the stability constants for the zwitterionic forms are much smaller and complex formation is negligible with the protonated species. In the case of the other amino acids the differences are less pronounced. The results are interpreted in terms of hydrogen bonding, steric effects and electrostatic interactions between the amino acid moiety and the rims of the cyclodextrins, in addition to the inclusion of the side chain, and are supported by ¹H and ¹³C NMR investigations on the systems containing l-phenylalanine and l-tyrosine. The differences between the complex formation constants of the l- and d-enantiomers do not exceed the limits of experimental error in most cases.     

β-Cyclodextrin functionalized gold nanoparticles: characterization and its analytical application for l-tyrosine

β-Cyclodextrin functionalized gold nanoparticles (β-CD-Au NPs) were synthesized and characterized. The interaction of β-CD-Au NPs with l-tyrosine (l-Tyr) is studied by fluorescence and absorption spectroscopy. β-CD-Au NPs was found to remarkably quench fluorescence of l-Tyr at 305 nm. Experimental conditions including media acidity, temperature and concentration of buffer were examined for the inclusion formation. Furthermore, fluorescence resonance energy transfer occurred with Föster radius 2.39 nm. Apparent binding constants and corresponding thermodynamic parameters at different temperatures were calculated by Stern–Volmer equation and thermodynamic formula, respectively. The binding constant declines with the increasing temperature illustrating static quenching exists and the binding l-Tyr on β-CD-Au NPs is a spontaneous molecular interaction process in which entropy and Gibbs free energy decreases. Hydrophobic interaction and van der Waals forces could be in dominant action in the formation of the complex. The concentration of l-Tyr is proportional to decrease of the fluorescence intensity in the range of 0.02–1.5 μM with the detection limit (S/N = 3) of 1.6 nM. There is little interference from the coexisting substances normally used as the pharmaceutical main compositions. The proposed method combined the unique physical and chemical properties of the gold core with the molecular recognition ability of β-CD and possessed high sensitivity and good selectivety. It has been successfully applied to determination of l-Tyr in compound amino acid injection.     

Activity and Synergistic Antimicrobial Activity Between Diketopiperazines Against Bacteria In Vitro

The aim of the present study was to determine the synergistic effects of diketopiperazines [cyclo-(l-Pro-l-Leu) (1), cyclo-(d-Pro-l-Leu) (2), and cyclo-(d-Pro-l-Tyr) (3)] purified from a Bacillus sp. N strain associated with entomopathogenic nematode Rhabditis (Oscheius) sp. on the growth of bacteria. The minimum inhibitory concentration and minimum bactericidal concentration of the diketopiperazines was compared with that of the standard antibiotics. The synergistic antibacterial activities of the combination of diketopiperazines against pathogenic bacteria were assessed using the checkerboard assay and time?Ckill methods. The results of the present study showed that the combination effects of diketopiperazines were predominately synergistic (FIC index <0.5). Furthermore, time?Ckill study showed that the growth of the tested bacteria was completely attenuated with 4?C12?h of treatment with 50:50 ratios of diketopiperazines. These results suggest that the combination of diketopiperazines may be microbiologically beneficial. The three diketopiperazines are nontoxic to normal human cell line (L231 lung epithelial) up to 200?m???g/ml. The in vitro synergistic activity of cyclo-(l-Pro-l-Leu), cyclo-(d-Pro-l-Leu), and cyclo-(d-Pro-l-Tyr) against bacteria is reported here for the first time. These findings have potential implications in delaying the development of resistance as the antibacterial effect is achieved with lower concentrations of both drugs (diketopiperazines).     

Application of Hydrazino Dinitrophenyl-Amino Acids as Chiral Derivatizing Reagents for Liquid Chromatographic Enantioresolution of Carbonyl Compounds

A new series of chiral derivatizing reagents (CDRs) consisting of five hydrazino dinitrophenyl (HDNP)-amino acids (CDR 1?C5) was prepared by a two-step synthesis procedure starting from 1,5-difluoro-2,4-dinitrobenzene (DFDNB). In the first step, five fluoro-dinitrophenyl (FDNP)-reagents, namely FDNP-l-Leu, FDNP-l-Val, FDNP-l-Phe, FDNP-l-Ala and FDNP-d-Phg were synthesized by substituting one of the fluorine atoms in DFDNB moiety with amino acids l-Leu, l-Val, l-Phe, l-Ala and d-Phg, respectively. In the following step, the remaining fluorine atom of the FDNP reagents was substituted with hydrazine hydrate to obtain five HDNP reagents (i.e. CDR 1?C5; HDNP-l-Leu, HDNP-l-Val, HDNP-l-Phe, HDNP-l-Ala and HDNP-d-Phg). These five CDRs were used for synthesis of diastereomers of six racemic carbonyl compounds which were resolved by high-performance liquid chromatography using C18 column and gradient eluting mixture of acetonitrile or methanol with triethylammonium phosphate buffer with UV detection at 348 nm. Microwave irradiation was used for synthesis of both the CDRs and the diastereomers. The newly synthesized CDRs were observed to be superior in comparison to their counterparts having amino acid amides as chiral auxiliaries in terms of cost effectiveness and providing better resolution of diastereomers. The method was validated for limit of detection, linearity, accuracy and precision.     

Solid state radiolysis of non-proteinaceous amino acids in vacuum: astrochemical implications

The analysis of the amino acids present in Murchison meteorite and in other carbonaceous chondrites has revealed the presence of 66 different amino acids. Only eight of these 66 amino acids are proteinaceous amino acids used by the present terrestrial biochemistry in protein synthesis, the other 58 amino acids are somewhat “rare” or unusual or even “unknown” for the current terrestrial biochemistry. For this reason in the present work a series of “uncommon” non-proteinaceous amino acids, namely, l-2-aminobutyric acid, R(?)-2-aminobutyric acid, 2-aminoisobutyric acid (or α-aminoisobutyric acid), l-norleucine, l-norvaline, l-β-leucine, l-β-homoalanine, l-β-homoglutamic acid, S(?)-α-methylvaline and dl-3-aminoisobutyric acid were radiolyzed in vacuum at 3.2 MGy a dose equivalent to that emitted in 1.05 × 10⁹ years from the radionuclide decay in the bulk of asteroids or comets. The residual amount of each amino acid under study remained after radiolysis was determined by differential scanning calorimetry in comparison to pristine samples. For optically active amino acids, the residual amount of each amino acid remained after radiolysis was also determined by optical rotatory dispersion spectroscopy and by polarimetry. With these analytical techniques it was possible to measure also the degree of radioracemization undergone by each amino acid after radiolysis. It was found that the non-proteinaceous amino acids in general do not show a higher radiation and radioracemization resistance in comparison to the common 20 proteinaceous amino acids studied previously. The unique exception is represented by α-aminoisobutyric acid which shows an extraordinary resistance to radiolysis since 96.6 % is recovered unchanged after 3.2 MGy. Curiously α-aminoisobutyric acid is the most abundant amino acid found in carbonaceous chondrites. In Murchison meteorite α-aminoisobutyric acid represents more than 20 % of the total 66 amino acids found in this meteorite.     

β-d-Xylosidase from Selenomonas ruminantium: Thermodynamics of Enzyme-Catalyzed and Noncatalyzed Reactions

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Temperature dependence for hydrolysis of 4-nitrophenyl-β-d-xylopyranoside (4NPX), 4-nitrophenyl-α-l-arabinofuranoside (4NPA), and 1,4-β-d-xylobiose (X2) was determined on and off (k _non) the enzyme at pH 5.3, which lies in the pH-independent region for k _cat and k _non. Rate enhancements (k _cat/k _non) for 4NPX, 4NPA, and X2 are 4.3?×?10¹¹, 2.4?×?10⁹, and 3.7?×?10¹², respectively, at 25 °C and increase with decreasing temperature. Relative parameters k _cat ^4NPX/k _cat ^4NPA, k _cat ^4NPX/k _cat ^X2, and (k _cat/K _m)^4NPX/(k _cat/K _m)^X2 increase and (k _cat/K _m)^4NPX/(k _cat/K _m)^4NPA, (1/K _m)^4NPX/(1/K _m)^4NPA, and (1/K _m)^4NPX/(1/K _m)^X2 decrease with increasing temperature.     

Improvement of the Production Efficiency of l-(+)-Tartaric Acid by Heterogeneous Whole-Cell Bioconversion

An Escherichia coli-engineered bacterium with cis-epoxysuccinate hydrolase (ESH) activity was used to catalyze the stereospecific hydrolysis of cis-epoxysuccinic acid to l-(+)-tartaric acid. The effect of the substrate composition on the production efficiency of l-(+)-tartaric acid was investigated. Based on the sodium-type homogeneous substrate system, a heterogeneous substrate system, composed of 1.2 M sodium-type substrate and 1.8 M calcium-type substrate, was designed to improve ESH catalytic efficiency. After process optimization, a catalytic efficiency of 9.37?×?10^?3 g U^?1 h^?1 was obtained with fed-batch mode in the heterogeneous substrate system, about a twofold increase compared to the traditional bioconversion process with Nocardia tartaricans cells. The scale-up tests were carried out in a 15-m³ stirred tank reactor, which indicated that the heterogeneous substrate system had great application prospect for the l-(+)-tartaric acid industrial production.     

Cis-Cycloprolylprolin-Anion-ein konfigurationsstabiles Carbanion

The racemisation ofcyclo-(l-Pro?l-Pro) (2) with metal amides in liq. ammonia was examined. The K-kation causes more extensive racemisation than Na-kation, which in turn is more effective than Li⁺. This, the racemisation of2 int-butyl alcohol with K⁺C₆H₅O^? and the data gained from corresponding deuterated medium show that the racemisation of2 proceeds in two steps: in the first, the less stabletrans-cyclo-(l-Pro?d-Pro) (3) is formed, followed by the rapid conversion of3 to a mixture ofcyclo-(l-Pro?l-Pro) andcyclo-(d-Pro?d-Pro) in the second step.     

Asteropsiside A and other asterosaponins from the starfish Asteropsis carinifera

Three asterosaponins were isolated from the tropical starfish Asteropsis carinifera: a new one, asteropsiside A, and two known ones, regularoside A and thornasteroside A. The structure of the new compound was established using 2D NMR spectroscopy and ESI mass spectrometry as the sodium salt of 3-O-sulfonato-(20E)-6-O-{β-d-fucopyranosyl-(1→2)-β-d-galactopyranosyl-(1→4)-[β-d-quinovopyranosyl-(1→2)]-β-d-xylopyranosyl-(1→3)-β-d-quinovopyranosyl}-3β,6α-dihydroxy-5α-cholesta-9(11),20(22)-dien-23-one. Regularoside A and thornasteroside A were shown to display the ability to inhibit the growth of the T-47D and RPMI-7951 tumor cell colonies in vitro.     

β-d-Xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-Site Inhibitor

β-d-Xylosidase/α-l-arabinofuranosidase from Selenomonas ruminantium is the most active enzyme known for catalyzing hydrolysis of 1,4-β-d-xylooligosaccharides to d-xylose. Catalysis and inhibitor binding by the GH43 β-xylosidase are governed by the protonation states of catalytic base (D14, pK _a 5.0) and catalytic acid (E186, pK _a 7.2). Biphasic inhibition by triethanolamine of E186A preparations reveals minor contamination by wild-type-like enzyme, the contaminant likely originating from translational misreading. Titration of E186A preparations with triethanolamine allows resolution of binding and kinetic parameters of the E186A mutant from those of the contaminant. The E186A mutation abolishes the pK _a assigned to E186; mutant enzyme binds only the neutral aminoalcohol $ \left( {{\text{pH}} - {\text{independent}}\;K_{\text{i}}^{\text{triethanolamine}} = 19\,{\text{mM}}} \right) $ , whereas wild-type enzyme binds only the cationic aminoalcohol $ \left( {{\text{pH}} - {\text{independent}}\;K_{\text{i}}^{\text{triethanolamine}} = 0.065\,{\text{mM}}} \right) $ . At pH 7.0 and 25°C, relative kinetic parameter, $ k_{\text{cat}}^{\text{4NPX}}/k_{\text{cat}}^{\text{4NPA}} $ , for substrates 4-nitrophenyl-β-d-xylopyranoside (4NPX) and 4-nitrophenyl-α-l-arabinofuranoside (4NPA) of E186A is 100-fold that of wild-type enzyme, consistent with the view that, on the enzyme, protonation is of greater importance to the transition state of 4NPA whereas ring deformation dominates the transition state of 4NPX.     

Exploiting thermodynamic data to optimize the enantioseparation of underivatized amino acids in ligand exchange capillary electrophoresis

Stereoselective amino acid analysis has increasingly moved into the scope of interest of the scientific community. In this work, we report a study on the chiral separation of underivatized d,l-His by ligand exchange capillary electrophoresis (LECE), utilizing accurate ex ante calculations. This has been obtained by the addition to the background electrolytes (BGE) of NaClO₄ which renders the separations “all in solution processes”, allowing to accurately calculate in advance the concentrations of the species present in solution and to optimize the system performances. To this aim, the formation of ternary complexes of Cu²⁺ ion and l-lysine (l-Lys) or l-ornithine (l-Orn) with l- and d-histidine (His), and histamine (Hm) have been studied by potentiometry and calorimetry at 25 °C and with 0.1 mol dm^?3 (KNO₃) in aqueous solution. The ternary species [Cu(L)(l-His)H]⁺ and [Cu(L)(d-His)H]⁺ (where L?=?l-Lys or l-Orn) show a slight but still detectable stereoselectivity, and the determination of ΔH° and ΔS° values allowed the understanding of the factors which determine this phenomenon. The stereoselectivity showed by the protonated ternary species has been exploited to chirally separate d,l-His in LECE, by using the binary complexes of copper(II) with l-Lys or l-Orn as background electrolytes added with the appropriate amounts of NaClO₄.